research article chemical exposure generates dna copy...

Research ArticleChemical Exposure Generates DNA Copy Number Variants andImpacts Gene Expression

Samuel M. Peterson1,2 and Jennifer L. Freeman1

1School of Health Sciences, Purdue University, 550 Stadium Mall Drive, HAMP-1263D, West Lafayette, IN 47907, USA2Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

Correspondence should be addressed to Jennifer L. Freeman; [email protected]

Received 29 August 2014; Revised 3 December 2014; Accepted 3 December 2014; Published 30 December 2014

Academic Editor: Mugimane Manjanatha

Copyright © 2014 S. M. Peterson and J. L. Freeman. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

DNA copy number variation is long associated with highly penetrant genomic disorders, but it was not until recently that thewidespread occurrence of copy number variation among phenotypically normal individuals was realized as a considerable sourceof genetic variation. It is also now appreciated that copy number variants (CNVs) play a role in the onset of complex diseases. Manyof the complex diseases in which CNVs are associated are reported to be influenced by yet to be identified environmental factors.It is hypothesized that exposure to environmental chemicals generates CNVs and influences disease onset and pathogenesis. Inthis study a proof of principle experiment was completed with ethyl methanesulfonate (EMS) and cytosine arabinoside (Ara-C)to investigate the generation of CNVs using array comparative genomic hybridization (CGH) and the zebrafish vertebrate modelsystem. Exposure to both chemicals resulted in CNVs. CNVs were detected in similar genomic regions among multiple exposureconcentrations with EMS and five CNVs were common among both chemicals. Furthermore, CNVs were correlated to alteredgene expression. This study suggests that chemical exposure generates CNVs with impacts on gene expression warranting furtherinvestigation of this phenomenon with environmental chemicals.

1. Introduction

Structural genetic variation in the human genome is presentin many forms including single nucleotide polymorphisms(SNPs), variable tandem repeats (e.g., mini- and microsatel-lites), presence/absence of transposable elements, and struc-tural alterations (e.g., deletions, duplications, and inversions).Until recently, SNPs were thought to be the predominantform of genomic variation and to account for much of thenormal phenotypic variation [1]. Recent developments andapplications of genome-wide technologies led to the discov-ery of thousands of copy number variants (CNVs) in thegenomes of phenotypically normal humans [2, 3]. CNVs aredefined as a duplication or deletion (i.e., a gain or loss ofa genomic DNA segment relative to a reference sample)measuring greater than 1 kb in size [4]. Human genomic copynumber variation has been studied for over 40 years, but itwas assumed that CNVs were few in number, had a rela-tively limited impact on the total amount of human geneticvariation, and were mainly associated with highly penetrant

disease phenotypes. In 2004, two studies independentlyreported the widespread presence of CNVs in the genomes ofphenotypically normal individuals [2, 3]. Following these ini-tial studies, additional genome-wide analyses identified andcharacterized novel human CNVs (e.g., [5]). Widespreadcopy number variation in the human genome is nowwell doc-umented with many CNVs spanning genes that are likely toaffect gene networks [6]. CNVs result in various phenotypiceffects including changes in gene expression levels, disruptionof gene dosage and regulatory elements, and loss of regulatoryelements [7].

Classical cytogenetics identified a variety of genomicvariants that are related to disorders that are caused by a singlevariant (e.g., a deletion on chromosome 7 inWilliams Beurensyndrome). CNVs that did not directly result in early-onset,highly penetrant genomic disorders were initially consideredneutral in function, but CNVs are now appreciated to play arole in the onset of complex diseases including autism spec-trum disorder (ASD), attention-deficit hyperactivity disorder

Hindawi Publishing CorporationAdvances in ToxicologyVolume 2014, Article ID 984319, 13 pageshttp://dx.doi.org/10.1155/2014/984319

2 Advances in Toxicology

(ADHD), and schizophrenia [8, 9]. In addition, CNVs arereported to influence late-onset diseases (e.g., Alzheimer’sdisease and Parkinson’s disease). In addition to geneticfactors, these diseases are also implicated to be influenced byenvironmental factors. The mechanisms by which environ-mental factors influence onset and pathogenesis of these dis-eases are not completely understood [10]. Current analysis offunctional attributes of CNV regions is revealing enrichmentfor genes that are relevant to molecular-environmental inter-actions [3, 5].Moreover, a study in postmortembrains of indi-viduals with ASD indicates possible involvement of exposureto polychlorinated biphenyls (PCBs) with a duplication eventon human chromosome 15 [11]. This study indicates an envi-ronmental link with CNVs and influence on complex disease,but it is not known if the copy number alteration was specif-ically generated by the environmental chemical exposure.

Exposure to environmental chemicals is one environ-mental factor that may contribute to the formation of CNVs(or copy number aberrations), but the ability of chemicalexposure to generate CNVs has not been thoroughly investi-gated.With the development of genomic technologies includ-ing the array comparative genomic hybridization (CGH)technology and NextGen sequencing, copy number alter-ations are now efficiently detected throughout the genome.Previous assays and techniques applied to investigate theinfluence of chemical exposure on the genomewere limited todetecting single nucleotide mutations or larger chromosomalaberrations (Figure 1). In addition, many of these assays hadinefficient integration of structural DNA alterations with thereference genome sequence limiting further studies into thebiological and functional significance of these DNA alter-ations. Thus, this class of DNA alteration was not thoroughlyassessed in past genotoxicity studies. Three recent studiesbegan to investigate the generation of CNVswith aphidicolin,hydroxyurea, and ionizing radiation in a cell culture system[12–14], but no other agents have been investigated to date.

The importance of using genomics to identify environ-mental chemical influence on the human genome is nowrecognized [15] and the specific influence of CNVs is recog-nized as an emerging environmental health issue (http://nas-sites.org/emergingscience/meetings/genomic-plasticity/). Inthis study, a proof of principle experiment using a zebrafishcell line was completed to test the hypothesis that chemicalexposure will result in CNVs detectable with the use ofarray CGH technology to set the stage for future analysisinto the influence of environmental chemical exposure ingenerating CNVs. The zebrafish is a prominent model verte-brate system in a variety of biological disciplines. A finishedgenome sequence and conserved genetic function betweenthe zebrafish and human genomes permit translation ofmolecular mechanisms of toxicity observed in the zebrafishmodel system to humans [16, 17]. Several zebrafish orthologsare reported to play a key role in human disease and large-scale mutant screens demonstrate that mutations in some ofthese orthologs display phenotypes similar to those presentin human diseases [18, 19]. In addition, the zebrafish hasbeen used for many years as a toxicological model [20] anda model for DNA repair mechanisms (e.g., [21]). A CNVmap is established for the zebrafish genome and confirms

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∙ Point mutations∙ Base pair substitutions∙ Frameshift mutations

∙ Microsatellites, minisatellites∙ Inversions

∙ Variable number tandem repeats

∙ Copy number alterations∙ Inversions∙ Translocations

∙ Chromosomal deletions∙ Chromosomal insertions∙ Chromosomal inversions∙ Intrachromosomal translocations

∙ Interchromosomal translocations∙ Ring chromosomes, isochromosomes∙ Aneuploidy∙ Polyploidy

∙ Di-, tri-, tetranucleotide repeats

2bp to 1,000 bp

Microscopic to subchromosomal

1kb to submicroscopic

Figure 1: Toxicity assays interrogating DNA sequence alterationsby size. Classic cytogenetic methodologies routinely identify wholegenome, whole chromosomal, and microscopic structural chromo-somal aberrations. At the opposite end of the spectrum, muta-tion assays are optimized to detect single base pair mutations.Development of genome-wide technologies including array-basedassays (e.g., array CGH) and whole genome sequencing now permitefficient detection of DNA structural alterations of an intermediatesize including copy number alterations and enable direct integrationwith a reference genome sequence. As a result, the ability of chemicalexposure to induce this type of DNA alteration was not thoroughlyinvestigated in the past and is just now beginning to be addressed.

the plasticity of the zebrafish genomepermitsCNV formation[22] and suitability for application in this study. Two geno-toxic chemicals commonly used as reference chemicals, ethylmethanesulfonate (EMS), and cytosine arabinoside (Ara-C)were included in this proof of principle experiment at mul-tiple exposure concentrations to assess dose-response anddifferences among the two chemicals. In addition, global geneexpression analysis was completed to correlate CNVs causedby chemical exposure and alterations in gene expression.

2. Materials and Methods

2.1. Cell Line and Toxicity Assay. A zebrafish fibroblast cellline established from approximately 100 embryos of theAB zebrafish strain that is described in detail in Freemanet al. [23] was used in this study. This zebrafish cell linewas used in this proof of principle study since it is well-characterized, is routinelymonitored for cytogenetic changes,and has been used in previous zebrafish cytogenetic studies[23]. In addition, use of this zebrafish cell line will provideease in moving into in vivo studies with in vivo zebrafish

Advances in Toxicology 3

in future studies. Two reference chemicals routinely used ingenotoxicity assays, ethyl methanesulfonate (EMS; CAS 62-50-0; Sigma, St. Louis, MO) and cytosine arabinoside (Ara-C; CAS 147-94-4; Sigma, St. Louis,MO), were investigated forpotential to generate CNVs. A cell confluency assay was firstcompleted to identify the toxicity of EMS and Ara-C in thiscell line. This assay is modified from Plewa et al. [24]. Briefly,cells were harvested from cell culture flasks following astandard trypsin protocol and cell concentration determined.The assay was set up in 96-well plates with 7,000 cells perwell in an appropriate volume of media and chemical stockto achieve desired chemical test concentrations. Plate set-upincluded a first column blank and a second column negativecontrol. Each plate contained four subsample wells perchemical concentration. Following set-up, plates were placedin an incubator at 28∘C and 5% CO

2. After 72 hours, the

cells were fixed in 50% methanol and stained with 1% crystalviolet in 50% methanol, and excess crystal violet solutionwas washed from the plate. The cells were then treated with1% SDS to bring the crystal violet back into solution. Theabsorbance of each well was read on a microplate readerat 595 nm and readings from the four subsample wells ofeach test concentration averaged. A percent negative controlvalue was calculated for each test concentration. This valuerepresents the confluency of the cells grown in the presenceof the test compound as compared to the unexposed controlcells. Three replicate plates were completed and the averagepercent negative control values of the three replicate plates foreach test concentration calculated, plotted, and fit a sigmoidalcurve. The 50% and 75% confluency value of the negativecontrol was calculated for each chemical to determine testconcentrations for array CGH analysis. These values werechosen to be able to compare if CNVs would be generatedfrom exposure treatments that ranged from 50% impactson cell confluency to exposure treatments that did not altercell confluency in the EMS experiment and to then chooseexposure treatments that did not impact cell confluency inthe Ara-C experiment.

2.2. Copy Number Analysis. A zebrafish-specific oligonu-cleotide platform was designed and printed in conjunctionwith Roche NimbleGen (Madison, WI) for this study toanalyze copy number changes. The zebrafish oligonucleotideplatform was manufactured using Roche NimbleGen’s pro-prietary Maskless Array Synthesizer technology using pho-tomediated synthesis chemistry. DNA probes were selectedusing a proprietary probe screening system. 𝑇

𝑚-balanced

probe selection was coupled with heuristic and Al predictivemethods derived from their experimental database. Probesets were selected to represent the genomic target andto have excellent hybridization characteristics. Specificallyfor this design, segmental duplications (i.e., regions of thegenome with up to 5 close matches) were included as somecopy number alterations are reported to be associated withthese genomic segments [5] and highly repetitive sequenceswere excluded. In addition, a number of standards werealso included throughout the array. A number of self toself-hybridizations were first conducted with this platform

to assess the performance of the array platform and todetermine resolution of platform.

ArrayCGHanalysis was performed similarly as describedin Peterson and Freeman [25]. Zebrafish cells were exposedto three chemical concentrations of EMS calculated from thecytotoxicity curve to represent (1) a concentration 50% of thenegative control value, (2) a concentration 75%of the negativecontrol value, and (3) a concentration where no cytotoxicimpacts were observed for a dose-response assessment and acorresponding negative control without chemical exposure.Two exposure concentrations with limited cytotoxicity wereincluded for Ara-C. Cells were harvested from maintenancecultures and cell concentration determined. Appropriatevolume of media and chemical stock was added to eachpetri dish to achieve the desired test concentrations (i.e.,0mM, 0.5mM, 2mM, and 5mM for EMS and 0𝜇M, 0.1 𝜇M,and 1 𝜇M for Ara-C). 7.5 × 105 cells were initially seededinto each dish. After set-up, petri dishes were placed in anincubator at 28∘C and 5% CO

2for 72 hours (the equivalent

of 1.5 cell cycle lengths). After 72 hours, cells were harvestedand genomic DNA was isolated following a standard phenol:chloroform isolation method as described in Freeman et al.[26]. Genomic DNA quantity and quality were determinedusing a NanoDrop ND-1000 and gel electrophoresis. Thenegative control was the reference sample for each replicateand was cohybridized with each treatment (i.e., the negativecontrol and the three treatment concentrations) using a two-color hybridization strategy. Genomic DNA samples werelabeled and subsequently hybridized upon the zebrafish arrayCGH platform using the protocol outlined in the RocheNimbleGen User’s Guide (Roche NimbleGen, Madison, WI).For each array hybridization, 1𝜇g of test DNA and 1 𝜇g ofreference DNA were fluorescently labeled with dye-labeled9mers (i.e., the test DNAs were labeled with Cy3 and thereference DNA was labeled with Cy5). Dye-incorporationand DNA quality and quantity were assessed using a Nan-oDrop ND-1000 spectrophotometer. Cy3-labeled test DNAand Cy5-labeled reference DNA was combined into one tubefor each test concentration and injected into a mixer attachedto the array CGH chip as described in the NimbleGen ArrayUser’s Guide. The chip was placed in a bay of the NimbleGenHybridization System and DNA hybridized for 16 hours at42∘C. Following hybridization, the arrays were washed insolutions supplied in the Roche NimbleGen wash buffer kitfollowed by spin drying of the slides in a microfuge slidedryer.

Hybridized arrays were scanned using two-color scan-ning for Cy3 and Cy5 at 5 microns on a GenePix 4000B(Molecular Devices, Sunnyvale, CA). Scans were optimizedfor Cy3 and Cy5 signal intensities in the same range andfor ∼1% of the features saturated. Array image data wasextracted using the NimbleScan software program (RocheNimbleGen, Madison, WI). The Cy3 and Cy5 signal inten-sities were normalized to one another using qspline nor-malization, a simple and robust nonlinear method of nor-malization for two-color experiments [27]. Normalized sig-nal intensity files were generated by NimbleScan. Internalcontrol probes and overall variation of signal intensity wereused to assess the quality of each array CGH experiment.


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Figure 2: Cell toxicity assay. The toxicity profile of (a) EMS and (b) Ara-C was first completed in the zebrafish cell line to determine impactson cell confluency in this specific cell line. From this analysis chemical concentrations were determined for array CGH experiments.

The NimbleScan data was then exported into the NexusCopy Number software to calculate DNA sequence regionsthat deviated from the expected 1 : 1 molar ratio of the testto reference DNA (log

2ratio of 0) similar to as reported

previously [22]. Called regions represent CNVs as a resultof chemical exposure. Genomic locations and magnitudeof gain/loss were compared among the experiments andamong the chemical treatments. Genomic locations of CNVswere integrated with the zebrafish reference sequence forcharacterization. Genomic location of CNVs was comparedamong samples and overlapping segments determined. Twoseparate experiments were completed for each chemical.

2.3. Global Gene Expression Analysis. To investigate theimpacts of CNVs caused by chemical exposure on geneexpression, global gene expression analysis was performedwith RNA isolated from a 2mM EMS exposure and acontrol treatment following similar procedures as describedpreviously [28]. The 2mM EMS treatment was chosen asan exposure that had a minor effect on confluency of thecells and at which a number of CNVs were detected. Threebiological replicates were included that consisted of threeseparate control samples and three separate samples treatedwith 2mM EMS. Microarray analysis was performed simi-larly to as described in Peterson et al. [28] with the zebrafish385K expression platform (Roche NimbleGen, Madison,WI) using the one-color hybridization strategy. As such,six different microarrays were hybridized for this analysis.This platform contains 385,000 60-mer probes interrogating37,157 targets with up to 12 probes per target. Followinghybridization, arrays were washed and scanned at 5 micronsusing a GenePix 4000B array scanner (Molecular Devices,Sunnyvale, CA). Array image data was extracted using

theNimbleScan software program (RocheNimbleGen,Madi-son, WI). Fluorescence signal intensities were normalizedusing quantile normalization [29] and gene calls generatedusing the Robust Multichip Average algorithm [30] fol-lowing manufacturer recommendations. Further statisticalprocessing of the array data was performed with ArrayStar (DNASTAR, Inc., Madison, WI) and Ingenuity PathwayAnalysis software (Ingenuity Systems, Redwood City, CA)to identify specific genes altered following EMS exposure.A robust and reproducible list of differentially expressedgenes using recommendations from the Microarray Qual-ity Consortium [31, 32] was first determined by genesconsistently expressed (Students 𝑡-test, 𝑃 < 0.05) andsubstantially altered with a fold change of ±2.0. Genomiclocation of genes with altered expressionwas compared to thegenomic location of CNVs and gene ontology analysis andmolecular pathway analysis completed using UCSC GenomeBrowser (http://www.genome.ucsc.edu/) and Ingenuity Path-way Analysis (IPA) software following similar parameters asin previous experiments [28]. All genes were converted andreported as human homologs.

3. Results

3.1. Cell Toxicity. The toxicity of EMS and Ara-C in thezebrafish cell line was first investigated and results wereused to determine the exposure concentrations for the arrayCGH analysis. Test concentrations were calculated at the 50%negative control value, at 75% negative control value, and ata concentration where no impacts on cell confluency wereobserved for a dose-response assessment in the array CGHanalysis for EMS (Figure 2(a)). 5mM, 2mM, and 0.5mM,were chosen as test concentrations for EMS, respectively.


Table 1: Genome coverage of the zebrafish array CGH platform.

Seq. IDNumber

ofprobes

Meaninterval(bp)

Medianinterval(bp)

Coverage(bp)

Chr1: 1–56204684 16932 3204 3208 956774Chr2: 1–54366722 16410 3204 3208 925430Chr3: 1–62931207 18916 3204 3209 1066623Chr4: 1–42602441 12659 3204 3209 711800Chr5: 1–70371393 21149 3204 3209 1192946Chr6: 1–59200669 17707 3204 3209 1000358Chr7: 1–70262009 21110 3204 3208 1193208Chr8: 1–56456705 16885 3204 3209 953452Chr9: 1–51490918 15560 3204 3208 879929Chr10: 1–42379582 12784 3204 3208 722041Chr11: 1–44616367 13512 3204 3209 763782Chr12: 1–47523734 14322 3204 3209 808732Chr13: 1–53547397 16177 3204 3208 913627Chr14: 1–56522864 16923 3205 3211 951969Chr15: 1–46629432 14027 3204 3209 790586Chr16: 1–53070661 16002 3204 3208 902893Chr17: 1–52310423 15830 3204 3208 893908Chr18: 1–49281368 14898 3204 3209 841550Chr19: 1–46181231 13923 3204 3209 786221Chr20: 1–56528676 16867 3204 3210 951071Chr21: 1–46057314 13802 3205 3210 778363Chr22: 1–38981829 11629 3204 3210 653771Chr23: 1–46388020 14049 3204 3208 792579Chr24: 1–40293347 12198 3204 3208 688239Chr25: 1–32876240 9860 3204 3209 554616Summary 384131 3204 3209 21674468

In addition, two concentrations with limited impacts tocell confluency were included for Ara-C (0.1 𝜇M and 1 𝜇M;Figure 2(b)).

3.2. CNVs following Chemical Exposure. The zebrafisholigonucleotide array CGH platform contains 385,000probes, approximately 50 to 75 nucleotides in length, tilingthe zebrafish genome with a median spacing of ∼3.2 kb(Table 1). Four self to self-hybridization experiments werefirst conducted to assess the performance of the platformand to determine the resolution of the platform. No callswere found to present in these self to self-hybridizationexperiments greater than 5 consecutive probes in length(∼12.8 kb) and the resolution of the platform was estimatedat 16 kb (6 consecutive probes in length). All oligonucleotidearray-based platforms have some degree of backgroundnoise, which varies among each specific platform. As aresult there is generally a lack of confidence in single probecalls. Evaluating a series of self to self-hybridizations (inwhich no copy number alterations should be observed)and confirmatory experiments for calls observed on thisplatform, we determined that calls containing at least

020406080

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Figure 3: Self to self-hybridization assessment. A series of four selfto self-hybridizations were conducted to assess the performance ofthe array CGH platform to determine the number of consecutiveprobes that are needed to have high confidence in a true call.From these experiments, it was determined that high confidence isattained in calls in which at least six consecutive probes significantlydeviate from the expected 1 : 1 molar ratio. As a result, resolution ofthis platform is approximately 16 kb.

6 consecutive probes have a high degree of confidence(Figure 3). Using these calling parameters, the number offalse positive calls was significantly decreased. In addition,calls had an average segmentation mean ±0.075 or greater inmagnitude.

For assessment of EMS, two separate experimentswere performed using the two-color hybridization strategy.Genomic DNA from each chemical treatment was cohy-bridized with the respective negative control as the referencesample. In the first experiment, 5, 17, and 1 CNVs were calledin the 0.5, 2, and 5mM treatments, respectively (Table 2). Inthe second experiment, 10, 0, and 11 CNVs were called inthe 0.5, 2, and 5mM treatments, respectively (Table 2). Intotal 44 CNVs were called with a loss in copy number in28 regions and a gain in copy number in 16 regions. CNVsranged from 19.8 to 7,069.8 kb in size. The number of CNVsdid not increase with dose, but there were 11 CNVs withoverlapping genomic locations among the EMS chemicaltreatments (Table 3). All overlapping CNVs agreed in theirrespective loss or gain in copy number furthering confidencein these calls. In addition, magnitude of change was alsosimilar among the overlapping CNVs. Overall 8 overlappingCNVs had a loss in copy number, while 3 overlappingCNVs had a gain in copy number. The overlapping genomicsegments were refined regions with a size ranging from33.5 to 1,283.8 kb. Three of the overlapping CNVs were inthree different samples including a loss on chromosome 4,a loss on chromosome 5, and a gain on chromosome 14(Figure 4), while the remaining were present in two samples(Table 3). Considering overlapping CNVs, a total of 29different copy number variable regions were present amongthe EMS treatments (Figure 5(a)). When CNVs generated byEMS exposure were compared to CNVs found in the ABstrain of zebrafish (the strain from which the cell line wasderived) 39% of the CNVs were found to overlap (Table 2).


Table 2: CNVs generated by EMS exposure.

Experiment Concentration (mM) Chromosome Start (bp) End (bp) Length (kb) Segmentation meanOverlapwith

CNVEsa

1 0.5 4 34804051 35101102 297.1 0.092 No1 0.5 8 12592572 13822847 1230.3 −0.088 Yes1 0.5 9 50187567 50303563 116.0 0.248 No1 0.5 14 16605821 16732619 126.8 0.276 Yes1 0.5 24 35425802 35674998 249.2 −0.173 No1 2 4 22583174 22620167 37.0 −0.792 No1 2 5 32488684 32820694 332.0 −0.222 No1 2 5 64239559 64293196 53.6 −0.391 Yes1 2 6 49556168 50808094 1251.9 −0.199 No1 2 7 48895436 48948526 53.1 0.583 Yes1 2 8 32921651 33404812 483.2 −0.321 No1 2 9 50190782 50306820 116.0 0.445 No1 2 13 14841216 14864686 23.5 0.705 No1 2 13 40036069 40492860 456.8 0.151 Yes1 2 14 16612394 16785422 173.0 0.361 Yes1 2 16 17135247 18061907 926.7 0.157 No1 2 18 31915173 33340460 1425.3 −0.235 Yes1 2 20 8220598 8240372 19.8 0.799 No1 2 20 16618414 16980789 362.4 0.196 No1 2 21 8967733 9185499 217.8 0.244 Yes1 2 21 21053248 21398259 345.0 −0.228 No1 2 25 31027622 31064013 36.4 −0.579 No1 5 14 16609009 16769181 160.2 0.282 Yes2 0.5 4 22586693 22620167 33.5 −0.739 No2 0.5 5 32662667 32817496 154.8 −0.441 No2 0.5 7 87 418056 418.0 −0.182 Yes2 0.5 8 12566432 13625424 1059.0 −0.138 No2 0.5 13 51315349 51444199 128.9 0.363 No2 0.5 14 27806152 34875942 7069.8 −0.079 Yes2 0.5 18 31506143 31908755 402.6 0.117 No2 0.5 18 43478909 43530599 51.7 −0.521 No2 0.5 19 8399336 10004277 1604.9 −0.109 No2 0.5 24 35472028 35709297 237.3 −0.231 No2 5 4 22586693 22624474 37.8 −0.905 No2 5 5 32481902 32817496 335.6 −0.398 No2 5 5 37581072 38022638 441.6 −0.226 No2 5 5 64165549 65694964 1529.4 −0.139 Yes2 5 6 49600941 50828098 1227.2 −0.268 No2 5 7 87 355049 355.0 −0.285 Yes2 5 8 22262700 23108696 846.0 −0.227 Yes2 5 13 51328104 51486986 158.9 0.482 No2 5 14 31449275 33739196 2289.9 −0.159 Yes2 5 18 27977948 31396054 3418.1 −0.161 Yes2 5 18 31911993 33198929 1286.9 −0.260 YesaCNVEs as called in AB strain zebrafish in [22].


Table 3: Common copy number variable regions among EMS treatments.

Genomic region of overlap Size (kb) Concentration (mM) CNV genomic region Length (kb) Segmentation mean

Chr4: 22586693–22620167 33.50.5 Chr4: 22586693–22620167 33.5 −0.7392 Chr4: 22583174–22620167 37.0 −0.7925 Chr4: 22586693–22624474 37.8 −0.905

Chr5: 32662667–32817496 154.80.5 Chr5: 32662667–32817496 154.8 −0.4412 Chr5: 32488684–32820694 332.0 −0.2225 Chr5: 32481902–32817496 335.6 −0.398

Chr5: 64239559–64293196 53.6 2 Chr5: 64239559–64293196 53.6 −0.3915 Chr5: 64165549–65694964 1529.4 −0.139

Chr6: 49600941–50808094 1207.2 2 Chr6: 49556168–50808094 1251.9 −0.1995 Chr6: 49600941–50828098 1227.2 −0.268

Chr7: 87–355049 355.0 0.5 Chr7: 87–418056 418.0 −0.1825 Chr7: 87–355049 355.0 −0.285

Chr8: 12592572–13625424 1032.9 0.5 Chr8: 12566432–13625424 1059.0 −0.1380.5 Chr8: 12592572–13822847 1230.3 −0.088

Chr9: 50190782–50303563 112.8 0.5 Chr9: 50187567–50303563 116.0 0.2482 Chr9: 50190782–50306820 116.0 0.445

Chr13: 51328104–51444199 116.1 0.5 Chr13: 51315349–51444199 128.9 0.3635 Chr13: 51328104–51486986 158.9 0.482

Chr14: 16612394–16732619 120.20.5 Chr14: 16605821–16732619 126.8 0.2762 Chr14: 16612394–16785422 173.0 0.3615 Chr14: 16609009–16769181 160.2 0.282

Chr18: 31915173–33198929 1283.8 2 Chr18: 31915173–33340460 1425.3 −0.2355 Chr18: 31911993–33198929 1286.9 −0.260

Chr24: 35472028–35674998 203.0 0.5 Chr24: 35425802–35674998 249.2 −0.1730.5 Chr24: 35472028–35709297 237.3 −0.231

Similar to the EMS experiments, two separate experi-ments were completed for Ara-C. In the first experiment, 1CNV was called in the 0.1 𝜇M treatment, while 2 CNVs werecalled in the 1𝜇M treatment. In the second experiment noCNVs were called in the 0.1𝜇M treatment and 15 CNVs werecalled in the 1𝜇M treatment (Table 4). Of the 18 total CNVs,5 were losses and 13 were gains in copy number and rangedfrom 28.9 to 1,505.2 kb in size (Figure 5(b)). When CNVsgenerated by Ara-C exposure were compared to CNVs foundin the AB strain of zebrafish 44% of the CNVs were foundto overlap (Table 4). There was no overlapping CNVs amongthe two concentrations or among the two experiments, but5 CNVs did overlap with CNVs in the EMS experiment(Table 5). The length of these CNVs in the Ara-C and EMStreatments was similar. Two CNV regions on chromosomes9 and 14 had consistent gains in both chemical treatments.Two CNV regions on chromosomes 5 and 6 had a loss in theEMS treatment and a gain in copy number following Ara-Ctreatment, while the final CNV region on chromosome 21 hada gain in the EMS treatment and a loss in theAra-C treatment.

3.3. Comparative Gene Expression Analysis. To elucidateimpacts of CNVs on gene expression, global gene expressionanalysis was completed with the 2mM EMS treatment.After removal of redundant probes and accounting for geneorthology a total of 1,146 genes were mapped with alteredexpression. 979 genes were downregulated and 167 genes

were upregulated (see Supplementary Table 1 available in sup-plementary material online at http://dx.doi.org/10.1155/2014/984319). Gene ontology and pathway analysis with IPAindicated enrichment with genes associatedwith diseases anddisorders,molecular and cellular functions, and physiologicalsystem development and function (Table 6).

59% of CNV regions (10/17 regions) resulted in a directimpact on gene expression for genes mapping within theCNVs. Five of the ten regions contained genes orthologousto human genes (Table 7).Three CNVswere correlated with asingle gene, while two CNVs were correlated with two genes.There were 86% positive associations (a copy number gainassociated with increased expression or a copy number losswith decreased expression) and 14% negative associations (again associated with decreased expression or a loss associatedwith increased expression).

4. Discussion

Current knowledge on the role of chemical exposure in thegeneration of CNVs is limited. In this study, we applied azebrafish array CGH platform to investigate this phenome-non. CNV identification in the zebrafish genomewas recentlycompleted and confirmed the plasticity of the zebrafishgenome permits CNV formation [22]. In this proof ofprinciple experiment a zebrafish cell line was initially usedto investigate CNV generation associated with chemical


1.6

1.2

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ratio

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ratio

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Figure 4: CNVs in common genomic locations among EMS treatments. Multiple CNVs mapped to common genomic locations followingthe EMS treatments including a gain in copy number in all three concentrations on chromosome 14. This overlapping genomic segmentwas 120.2 kb in size and mapped to base pair region 16,612,394–16,732,619 on chromosome 14. (a) 0.5mM, (b) 2mM, and (c) 5mM EMStreatments.

Chr1

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(b)

Figure 5: CNVs generated by chemical exposure. (a) Exposure to EMS resulted in 44 CNVs among all treatments and consisted of 11 CNVswith overlapping genomic locations. Considering genomic location overlap, 29 different copy number variable regions were identified andincluded both gains (12; green bars) and losses (17; red bars). Length of bar indicates frequency. (b) Ara-C exposure resulted in 18 CNVsamong the treatments including 13 gains (green bars) and 5 losses (red bars). There were no overlapping CNVs among the Ara-C treatments.

exposure with the intention to translate these findings infuture in vivo studies using this model.

Two genotoxic chemicals, EMS and Ara-C, were testedfor their ability to generate CNVs. EMS is routinely used asreference chemical in genotoxicity assays that is reported to

directly produce random point mutations in genetic materialby direct alkylation and is oftenused as a chemicalmutagen instudieswithmodel organisms (e.g., [33]). Although alkylatingagents are thought to primarily generate point mutations,EMS is also reported to cause other genetic alterations


Table 4: CNVs generated by Ara-C exposure.

Experiment Concentration (𝜇M) Chromosome Start (bp) End (bp) Length (kb) Segmentation meanOverlapwith

CNVEsa

1 0.1 6 51918902 51957898 39.0 −0.443 No1 1 4 41297060 41404074 107.0 −0.298 Yes1 1 19 3146476 3308498 162.0 −0.206 Yes2 1 4 6479244 7984418 1505.2 0.104 Yes2 1 5 28310743 28556303 245.6 0.158 No2 1 5 32149703 32834420 684.7 0.144 Yes2 1 5 37513893 38141429 627.5 0.117 No2 1 6 33587936 33807346 219.4 0.196 No2 1 6 49575981 50700812 1124.8 0.126 No2 1 8 46328539 46502625 174.1 −0.237 Yes2 1 9 50187567 50306820 119.3 0.460 No2 1 12 11689110 12180785 491.7 0.173 Yes2 1 14 16605821 16765982 160.2 0.473 Yes2 1 17 39893636 39923656 30.0 0.455 No2 1 19 37183601 37212491 28.9 0.559 No2 1 20 3298313 3486787 188.5 0.226 No2 1 21 8953651 9198587 244.9 −0.194 Yes2 1 23 3153298 3185448 32.2 0.374 NoaCNVEs as called in AB strain zebrafish in [22].

Table 5: Common copy number variable regions in EMS and Ara-C treatments.

Chemical Concentration Chromosome Start (bp) End (bp) Length (kb) Segmentation meanEMS 5mM 5 37581072 38022638 441.6 −0.226Ara-C 1 𝜇M 5 37513893 38141429 627.5 0.117EMS 2mM 6 49556168 50808094 1251.9 −0.199EMS 5mM 6 49600941 50828098 1227.2 −0.268Ara-C 1 𝜇M 6 49575981 50700812 1124.8 0.126EMS 0.5mM 9 50187567 50303563 116.0 0.248EMS 2mM 9 50190782 50306820 116.0 0.445Ara-C 1 𝜇M 9 50187567 50306820 119.3 0.460EMS 0.5mM 14 16605821 16732619 126.8 0.276EMS 2mM 14 16612394 16785422 173.0 0.361EMS 5mM 14 16609009 16769181 160.2 0.282Ara-C 1 𝜇M 14 16605821 16765982 160.2 0.473EMS 2mM 21 8967733 9185499 217.8 0.244Ara-C 1 𝜇M 21 8953651 9198587 244.9 −0.194

including DNA strand breaks [34, 35]. A range of EMSchemical treatments from those that resulted in a 50%decrease in cell confluency to no impacts on cell confluencywere included in this experiment. An increase in the numberof CNVs was not observed with increasing dose, but CNVswere detected in similar genomic regions among the multipletest concentrations of EMS indicating a potential hotspot ofgenomic instability and a nonrandom genotoxic mechanismfor this chemical. Moreover, 39% of the CNVs generated byEMS exposure overlapped with known CNVs in the genomeof the AB strain of zebrafish [22] indicating these regionsmay

be more susceptible to genomic rearrangements than otherregions. Each of these experiments was started from the samebatch of cells with similar cytogenetic structure to alleviatedetection of background CNVs and thus support CNVsobserved in this study were due to the chemical exposure.

Ara-C was included as a comparative chemical to assessif CNVs are chemical-specific and to further assess CNVsat concentrations with limited alterations on cell confluency.Ara-C is also often used as a reference chemical in genotox-icity assays, is a chemotherapy agent, interferes with DNAsynthesis, and results in chromosomal aberrations [36, 37].


Table 6: Gene ontology analysis for enrichment of biologicalfunction with 2mM EMS treatment.

Biological function 𝑃 valuea Number ofgenesb

Diseases and disordersDevelopmental disorder 1.66𝐸 − 08 − 1.24𝐸 − 02 177Skeletal and musculardisorders 2.35𝐸 − 05 − 1.02𝐸 − 02 66

Infectious disease 9.31𝐸 − 05 − 1.04𝐸 − 02 170Connective tissuedisorder 1.03𝐸 − 04 − 6.50𝐸 − 03 40

Cardiovascular disease 1.27𝐸 − 04 − 7.41𝐸 − 03 43Molecular and cellularfunctions

Cellular movement 7.52𝐸 − 07 − 1.34𝐸 − 02 204Amino acid metabolism 3.36𝐸 − 06 − 1.14𝐸 − 02 78Small moleculebiochemistry 3.36𝐸 − 06 − 1.39𝐸 − 02 183

Cellular assembly andorganization 5.52𝐸 − 06 − 1.39𝐸 − 02 179

Cellular function andmaintenance 5.52𝐸 − 06 − 1.39𝐸 − 02 189

Physiological systemdevelopment and function

Tissue morphology 3.17𝐸 − 09 − 1.34𝐸 − 02 209Organismal survival 4.22𝐸 − 08 − 9.58𝐸 − 03 187Embryonic development 2.67𝐸 − 07 − 1.34𝐸 − 02 221Organismal development 3.24𝐸 − 06 − 1.35𝐸 − 02 284Organ morphology 1.29𝐸 − 05 − 1.17𝐸 − 02 143

aDerived from the likelihood of observing the degree of enrichment in a geneset of a given size by chance alone. Amaximum false discovery rate of 5%wasaccepted in this analysis.bClassified as being differentially expressed that relate to the specifiedfunction category; a gene may be present in more than one category.

In the Ara-C treatments, a higher number of CNVs weregenerated at the higher exposure treatment and no overlap-ping CNVs were detected between the two treatments, but44% of the CNVs generated by Ara-C exposure overlappedwith CNVs in the genome of the AB strain of zebrafish [22].While a number of chemical-specific CNVs were called, 5CNVs were generated in similar genomic regions in both theEMS and Ara-C treatments indicating these regions may bemore susceptible to genomic alterations in a nonchemical-specific manner. Two of the five regions were consistent ingain and/or loss in copy number in the specific genomicregion. These findings are similar to the patterns of CNVsgenerated by exposure to aphidicolin and hydroxyurea in astudy where CNVs were distributed among the genome withsome hotspots of formation [13].

Overall a dose-response was observed with the twotreatments of Ara-C, but not among the EMS treatments. Itis hypothesized that this difference is due to the effects oncell confluency among the two ranges of test concentrationsin the EMS versus the Ara-C experiments (i.e., the EMSexposure treatments ranged from those that resulted in 50%

cell confluency to no impact on cell confluency, while both ofthe Ara-C treatments did not impact cell confluency).

To further assess the influence of CNVs generated bychemical exposure, global gene expression analysis wasconducted for the 2mM EMS treatment. Overall geneexpression alterations were identified to be associated withdevelopmental disorders, skeletal muscular disorders, infec-tious diseases, connective tissue disorder, and cardiovasculardisease. In addition, alterations were associated with genesinvolved in cellularmovement, amino acidmetabolism, smallmolecular biochemistry, cellular assembly and organization,and cellular function and maintenance. Expression alter-ations were also enriched for genes associated with tissuemorphology, organismal survival, embryonic development,organismal development, and organ morphology. Further-more, a direct comparison of genomic regions harboringCNVs with the genomic location of genes with alteredexpression in the 2mM EMS treatment indicates that CNVsgenerated by chemical exposure impact gene expression.This analysis identified both direct associations and negativeassociations. The negative association may be regulatory innature. Genes with altered expression associated with CNVsinclude genes involved in SNAP receptor activity (STX16),the initiation of transcription (MED12), the initiation ofprotein synthesis (EIF2S), acetyl-CoA transport (SLC33A1),the de novo synthesis of purine nucleotides (GMPS), DNAbinding (ARID5B), and signal transduction (CAPN5). Genesare also associated with various diseases including a deletionin STX16 with autosomal dominant pseudohypothyroidism[38], a decrease in expression of EIF2S with uveal melanoma[39], a polymorphism in ARID5B with an increased risk ofMLL rearrangements in early childhood leukemia [40], andmutations in CAPN5 with autosomal dominant neovascularinflammatory vitreoretinopathy [41]. In addition, ARID5Bis essential for adipogenesis and liver development, whileCAPN5 plays an important role in developmental processes[42]. Moreover, it is likely the CNVs are also linked to alteredexpression of other genes as CNVs are reported to have globalinfluence on the transcriptome [43]. It should also be recog-nized that exposure to the genotoxic chemicals can result insingle nucleotide mutations. Thus, some of the detected geneexpression changesmay be due to single nucleotidemutationsand/or other DNA alterations, but overall the data indicatesthat theCNVs generated by the chemical treatments are likelyto contribute to gene expression changes and support furtherstudies in the functional effects of the CNVs generated by thechemical exposures.

The zebrafish genome is reported to have gone throughtwo rounds of whole genome duplication during the courseof evolution with a third event occurring before the lastteleost radiation [44]. The duplicated genome did lead tosome difficulty inmapping of the zebrafish genome comparedto the rodent and human genomes, but a finished referencesequence is now available [17]. These duplication events mayinfluence the presence of segmental duplications and is sug-gested to lead to an increase in CNVs in the zebrafish genome[22]. As such, the genome duplication may also influence thefrequency at which CNVs generated by chemical exposuresmay be observed. Additional studies will be needed with


Table7:Genes

with

alteredexpressio

nmapping

with

inCN

Vsinthe2

mM

EMStre

atment.

CNVregion

Genee

xpressionarraydata

Chromosom

eGenom

icregion

Gain/loss

Chromosom

eGenom

icregion

Sequ

ence

IDFo

ldchange

Hum

angene

Genes

ymbo

l

649556168–50808094

Loss

649674955–4

9688887

ENSD

ART

00000002693−5.037

Syntaxin

16ST

X16

649947447–4

9963592

BC06

6706.1

−2.129

Eukaryotictransla

tioninitiationfactor

2,subu

nit2

beta

EIF2S

832921651–33404

812

Loss

832982721–32982807

NM

001039461

−6.194

Mediatorc

omplex

subu

nit12

MED

12

1831915173–33340

460

Loss

1832385597–32398337

NM

20110

8−5.699

Solutecarrierfam

ily33

(acetyl-C

oAtransporter),m

ember1

SLC3

3A1

183240

8894–32442474

NM

200587

−13.404

Guanine

mon

opho

sphatesynthetase

GMPS

218967733–9185499

Gain

219178136–

9675872

ENSD

ART

0000

0054092−9.2

99AT

richinteractived

omain5B

(MRF

1-like)

ARID

5B

2121053248–21398259

Loss

2121135672–2116

6560

NM

001080007

−3.236

Calpain5

CAPN

5


other model systems to investigate and further understandthe influence of chemical exposure on generating CNVs.

5. Conclusions

Overall this study indicates that chemical exposure resultsin CNVs that alter gene expression. This study is setting thestage for future investigations into the specific mechanism offormation and expansion to assess environmental chemicals,additional structural genomic alterations using sequencingtechnologies, and inclusion of in vivo systems to study thebiological and functional significance of the CNVs and theirinfluence on disease pathways.

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper.

Acknowledgments

This work was supported by a Purdue Research Founda-tion Faculty Grant (JLF), a Purdue OVPR Incentive GrantProgram: Encouraging and Stimulating Purdue’s EmergingResearch: Category II Grant (JLF), and a Purdue ResearchFoundation Research Grant X1 (JLF, SMP).

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